Endonuclease V [EC 3.1.21.7] is an enzyme that may be referred to as deoxyinosine 3′-endonuclease, and this recognizes the base (hypoxanthine) of deoxyinosine in a DNA strand and hydrolyzes the phosphodiester bond (mainly the 2nd phosphodiester bond on the 3′-side of the recognized base) around it. In addition to the deoxyinosine-specific cleavage activity thereof, this enzyme recognizes a broad variety of DNA structures including deoxyuridine base (uracil), AP site (apurinic/apyrimidinic site or abasic site), base mismatches, base insertions/deletions, flap structures, pseudo-Y structures in DNA strands, therefore having activities of cleaving various DNA strands. Further, the enzyme has a nonspecific nucleic acid cleavage activity. Endonuclease V or a gene coding for it has been found in many organism species of organisms or isolated, and in particular, those derived from Escherichia coli and Thermotoga maritima have been relatively precisely investigated for their properties (Non-Patent References 1 to 11).
The enzymatic activities of endonuclease V are applicable to various inspection technologies, diagnostic technologies and gene-engineering technologies including analysis, detection, degradation, synthesis and modification of nucleic acid molecules (for example, Non-Patent References 12 to 16, and Patent References 1 to 12).
However, as so mentioned above, endonuclease V has not only a specific activity but also a nonspecific nucleic acid cleavage activity. For example, except for the case where it is used for random nicking of DNA strands (for example, Patent Reference 7), the nonspecific cleavage activity is a cause of bringing about an unfavorable result in almost all applications where the specific activity of the enzyme is utilized. Accordingly, the nonspecific nucleic acid cleavage activity of endonuclease V is an essential bar to the industrial applicability of this enzyme.
For overcoming the problem, for example, a method is disclosed, which comprises recombining the DNA strands that have been nonspecifically cleaved by endonuclease, by the use of a ligase (for example, Non-Patent References 13 to 15, Patent Reference 9).
However, no report has heretofore been made in the art relating to a method of essentially solving the problem itself in that endonuclease V has a nonspecific nucleic acid cleavage activity.
Patent Reference 1: WO 2004/003232
Patent Reference 2: WO 02/36821
Patent Reference 3: WO 2004/067765
Patent Reference 4: WO 2004/067726
Patent Reference 5: WO 2004/022701
Patent Reference 6: WO 03/080645
Patent Reference 7: WO 2004/046383
Patent Reference 8: WO 99/42595
Patent Reference 9: WO 02/44335
Patent Reference 10: US patent publication 2005/136417
Patent Reference 11: US patent publication 2005/123956
Patent Reference 12: WO2004/015105
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Non-Patent Reference 2: Yao M, Kow Y W: Strand-specific cleavage of mismatch-containing DNA by deoxyinosine 3′-endonuclease from Escherichia coli. J Biol Chem, 269, 31390-6 (1994).
Non-Patent Reference 3: Yao M, Kow Y W: Interaction of deoxyinosine 3′-endonuclease from Escherichia coli with DNA containing deoxyinosine. J Biol Chem, 270, 28609-16 (1995).
Non-Patent Reference 4: Yao M, Kow Y W: Cleavage of insertion/deletion mismatches, flap and pseudo-Y DNA structures by deoxyinosine 3′-endonuclease from Escherichia coli. J Biol Chem, 271, 30672-6 (1996).
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Non-Patent Reference 6: Zvonimir Siljkovic Crystal structure of the DNA repair enzyme endonuclease V from Thermotoga maritima. Master's Thesis, Purdue University, Thesis 46615 MS (2000).
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Non-Patent Reference 9: Liu J, He B, Qing H, Kow Y W: A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V. Mutat Res, 461, 169-77 (2000).
Non-Patent Reference 10: Hitchcock T M, Gao H, Cao: Cleavage of deoxyoxanosine-containing oligodeoxyribonucleotides by bacterial endonuclease V. Nucleic Acids Res, 32, 4071-80 (2004).
Non-Patent Reference 11: Feng H, Klutz A M, Cao W Active Site Plasticity of Endonuclease V from Salmonella typhimurium. Biochemistry, 44, 675-83 (2005)
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Non-Patent Reference 13: Huang J, Kirk B, Favis R, Soussi T, Paty P, Cao W, Barany F: An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissue. oncogene, 21, 1909-21 (2002).
Non-Patent Reference 14: Favis R, Huang J, Gerry N P, Culliford A, Paty P, Soussi T, Barany F: Harmonized microarray/mutation scanning analysis of TP53 mutations in undissected colorectal tumors. Hum Mutat, 24, 63-75 (2004).
Non-Patent Reference 15: Pincas H, Pingle M R, Huang J, Lao K, Paty P B, Friedman A M, Barany F: High sensitivity EndoV mutation scanning through real-time ligase proofreading. Nucleic Acids Res. 32, e148 (2004).
Non-Patent Reference 16: Miyazaki K: Random DNA fragmentation with endonuclease V: application to DNA shuffling. Nucleic Acids Res, 30, e139 (2002).